For decades optical microscopy has been the workhorse of various fields including engineering, physical sciences, medicine and biology. Despite its long history, until relatively recently, there has not been a significant change in the design and working principles of optical microscopes. Over the last decade, motivated partially by the quest to better understand the realm of the nano-world, super-resolution techniques started a renaissance for optical microscopy by addressing some of the most fundamental limitations of optical imaging such as the diffraction limit. Besides these super-resolution techniques, several other novel imaging architectures were also implemented to improve the state of the art in optical microscopy towards better speed, signal to noise ratio (SNR), contrast, throughput, specificity, etc. This recent progress in microscopy utilized various innovative technologies to overcome the fundamental barriers in imaging and has created significant excitement in a diverse set of fields by enabling new discoveries to be made.
However, together with this progress, the overall complexity and the cost of the optical imaging platforms has increased. Expensive and sometimes large optical imaging systems often limit the widespread use of some of these advanced optical imaging modalities beyond well-equipped laboratories.
In the meantime, a rapid advancement in digital technologies has occurred, with much cheaper two-dimensional solid state detector arrays having significantly larger areas with smaller pixels, better dynamic ranges, frame rates and signal to noise ratios, as well as much faster, cheaper and more powerful digital processors and memories. This on-going digital revolution, when combined with advanced imaging theories and numerical algorithms, also creates an opportunity for optical imaging and microscopy to face another dimension in this renaissance towards simplification of the optical imaging apparatus, making it significantly more compact, cost-effective and easy to use, potentially without a trade-off in its performance.
Lenses for decades have been helping detectors (analog or digital) to operate at the lowest possible space-bandwidth product that is determined by the desired field-of-view and the resolution of the image. However, the above discussed digital revolution has already advanced the state of the art for digital imagers such that a 2D space-bandwidth product of >10-20 Million is readily available nowadays. This implies that today's detector arrays are now much better suited to handle the information distortion caused by diffraction, which may then raise questions on the absolute necessity of the use of lenses in optical imaging. Moreover, today's digital processors together with novel algorithms are also in much better shape to process, almost instantaneously, the acquired information at the detector end for taking the job of a physical lens. Looking at this picture, one can conclude that the widespread use of lenses (or similar wavefront shaping elements) in optical imaging can now be potentially replaced for several application needs (specifically for cell microscopy) by cost-effective, compact and much simpler optical architectures that compensate in the digital domain for the lack of complexity of optical components. This approach should especially address the needs and the requirements of resource limited settings, potentially providing a leapfrog in the fight against various global health related problems involving infectious diseases.
Quite importantly, microscopy in resource-limited settings has requirements considerably different from those of advanced laboratories, and such imaging devices should be simple to use and operate, cost-effective, compact, and light-weight, while at the same time being properly accurate. Another field that would enormously benefit from lensfree, compact and cost-effective on-chip digital imagers is the field of microfluidics. Over the last decade, microfluidics has revolutionized the available toolset to handle cells by significantly reducing the required device and reagent volumes as well as the associated costs. This has, in some instances, enabled so-called lab-on-a-chip applications. Despite all the progress that has occurred on merging optical technologies with microfluidics, one area that still remains relatively low-throughput, bulky and costly is the integration of optical microscopy platforms with microfluidic features found on such devices. Without significant miniaturization and simplification of this imaging platform together with an increase in throughput, the true extent of the microfluidic revolution cannot be fully realized especially for cytometry applications.
The fruits of this thinking have already appeared in the literature, where various lensfree on-chip imaging architectures were successfully demonstrated. See e.g., Xu, W., Jericho, M. H., Meinertzhagen, I. A. & Kruezer, H. J. Digital in-line holography for biological applications. Proc. Natl. Acad. Sci. U.S.A. 98, 11301-11305 (2001). Among these approaches, lensfree digital holography deserves a special attention since with new computational algorithms and mathematical models, it has the potential to make the most out of this digital revolution. In this context, lensfree digital in-line holography has already been successfully demonstrated for high-resolution microscopy of cells and other micro-organisms as described in Xu et al. above. Conventional coherent lensfree in-line holography approaches, however, demand near-perfect spatial coherence for illumination, and therefore require focusing of a laser light on a small aperture that is sized on the order of a wavelength for spatial filtering. The use of a small aperture size (e.g., 1-2 μm) requires a mechanically stable and a carefully aligned system together with a focusing lens to efficiently couple the laser radiation to the aperture for improved light throughput. This can require a robust system to ensure properly optical alignment and mechanical stability. In addition, keeping such a small aperture clean and operational over an extended period of time can be another challenge especially for uses outside the laboratory environment.
Further, in conventional lensfree in-line holography the cells of interest are typically positioned far away (e.g., >1-2 cm) from the sensor surface such that the holographic signature of each cell is spread substantially over the entire sensor area, where all the cells' particular holographic “signatures” significantly overlap. Such an approach unfortunately limits the imaging field-of-view (FOV) at the cell plane. All these requirements increase the cost and the size of the optical instrument. Further, these constraints also make conventional lensfree coherent in-line holography approaches inconvenient for use in resource-limited settings such as in the field.
Incoherent or partially coherent sources in holography have also been utilized in different lens-based optical architectures. These holographic imaging techniques are not, however, classified as “on-chip” as they utilize various bulky optical components and therefore they can be considered under the same category as the advanced imaging modalities discussed above making them much less suitable for uses outside a laboratory. Much simpler approaches using partially coherent lensfree in-line holography have also been recently demonstrated for imaging of latex particles, but these techniques also suffer from a small field-of-view as they position the objects-of-interest far away from the sensor surface. See e.g., Dubois, F., Requena, M. N., Minetti, C., Monnom, O. & Istasse, E. Partial spatial coherence effects in digital holographic microscopy with a laser source. Appl. Opt. 43, 1131-1139 (2004). Further, these studies used coupling optics for the illumination such as a microscope objective-lens and had relatively coarse imaging performance.